Transcription Associated Recombination in Mammalian Cells

Abstract: There is increasing evidence that the movement of the transcription machinery through DNA has profound effects on the genomic stability. One such example is a phenomenon known as Transcription Associated Recombination (TAR). Transcription enhances recombination levels to a high degree in all organisms studied, from bacteria to mammals. The underlying causes of the high recombination levels observed are unknown, as are the rationale for the rather riskyhazardous recombination event in this context. Recombination is not a risk-free event; there is e.g. the chancerisk for of loss of heterozygozity, which may eventually lead to tumour formation. So, why is TAR so ubiquitous? This thesis deals with the factors inducing TAR, trying to elucidate the mechanisms catalyzing this event. The proteins involved in executing TAR are unknown in mammals, and one of the aims of this thesis havehas been to investigate the role of well-known DNA repair proteins in TAR. In order to do so, cell lines deficient in crucial DNA repair proteins were stably transfected with a novel recombination construct. Transcription can be controlled over this recombination construct, enabling the detection of transcription associated recombination. We found that TAR is dependent on replication and that inhibition of transcription elongation had no further effect on TAR levels in our system. Further, we found that TAR employs a recombination pathway mechanistically separate from the recombination pathway induced by DNA double strand breaks. This pathway is dependent on BRCA2, a protein required for homologous recombination, but independent of the RAD51 paralog XRCC2. In subsequent studies, we found that the XPD subunit of the combined transcription and repair factor TFIIH is required for TAR, but is dispensable for DNA DSB repair by HR. We went on to investigate the connection between HR repair of UV damages and transcription and found that repair of UV damages requires transcription, but not via the transcription-coupled repair pathway. In conclusion, we found that TAR operates by a recombination pathway separate from DNA double strand break induced recombination. We found a connection with stalled replication, and revealed several of the proteins required for TAR in mammals.